should I go on to the ligation? urgent - (Mar/25/2007 )

Hi everyone.

I'm trying to clone an 500bp insert into pet28b vector by using Nhe1 and BamH1 as restriction enzyme. First i digested the vector and insert by Nhe1 overnight. This morning, I did BamH1 digestion for four hours after the PCR purification. But when I see the gel result for this digestion. I found both my vector and insert were degraded. But I still have some left running on the right positions for both vector and insert. I've cut the gel and put them in the 4 degree. I wonder in this case can I continue to the ligation step? the amount is not that much for the vector. Too sad right now.

I also run some sample after the Nhe1 digestion for both vector and insert, and they are fine. What would cause the degradation for bamH1 step??

Many thanksyouyou

-youyou-

I found using BamHI if you cut for too long time, it will have this problem. I usually cut the vector for 30min, and PCR product for 1 hour (because the amount is larger than vector). I think 4 hours is too long for it.

I am not sure whether you can go for the ligation step, but I will give it a try. And at the same time, you can try to cut the material again with shorter time.

QUOTE (youyou @ Mar 26 2007, 07:12 AM)

Hi everyone.

I'm trying to clone an 500bp insert into pet28b vector by using Nhe1 and BamH1 as restriction enzyme. First i digested the vector and insert by Nhe1 overnight. This morning, I did BamH1 digestion for four hours after the PCR purification. But when I see the gel result for this digestion. I found both my vector and insert were degraded. But I still have some left running on the right positions for both vector and insert. I've cut the gel and put them in the 4 degree. I wonder in this case can I continue to the ligation step? the amount is not that much for the vector. Too sad right now.

I also run some sample after the Nhe1 digestion for both vector and insert, and they are fine. What would cause the degradation for bamH1 step??

Many thanksyouyou

-Sorva-

thanks a lot. I'll try it.

QUOTE (Sorva @ Mar 25 2007, 05:43 PM)

I found using BamHI if you cut for too long time, it will have this problem. I usually cut the vector for 30min, and PCR product for 1 hour (because the amount is larger than vector). I think 4 hours is too long for it.

I am not sure whether you can go for the ligation step, but I will give it a try. And at the same time, you can try to cut the material again with shorter time.

QUOTE (youyou @ Mar 26 2007, 07:12 AM)

Hi everyone.

I'm trying to clone an 500bp insert into pet28b vector by using Nhe1 and BamH1 as restriction enzyme. First i digested the vector and insert by Nhe1 overnight. This morning, I did BamH1 digestion for four hours after the PCR purification. But when I see the gel result for this digestion. I found both my vector and insert were degraded. But I still have some left running on the right positions for both vector and insert. I've cut the gel and put them in the 4 degree. I wonder in this case can I continue to the ligation step? the amount is not that much for the vector. Too sad right now.

I also run some sample after the Nhe1 digestion for both vector and insert, and they are fine. What would cause the degradation for bamH1 step??

Many thanksyouyou

-youyou-

another question, previously BamH1's buffer is always BamH1 buffer, why this time the new enzyme i ordered came in with the NEbuffer 3?? They are not using the specific buffer for BamH1 any more? May this be the problem??

And how much enzyme should I used for 1ug DNA incubating 1 hour to avoid the star activity?

-youyou-

If you have both insert and vector of the correct size, you might as well try the ligation expt. If it works, you're a hero ligation expert; if it doesn't, it was a long shot, anyway! If the ligation doesn't work, may I suggest you cut down your BamHI digestion. Look at the definition of a unit: 1 ug of lambda (which has quite a few BamHI sites in each molecule) in 1 hr digestion. Unless you have a huge amount of DNA and very little enzyme, you are overdigesting by a long way (although, by 4 hrs, most of the enzyme is dead). Bam is also one of those enzymes that is very sensitive to its conditions. Use only what the data sheet recommends (1 units or 2 should be all you need, especially for a plasmid with only a single Bam site), don't go over 10% volume as enzyme, and keep the incubation time to 1 hr.